RNA-Seq Transcriptome Profiling Identifies CRISPLD2 As a Glucocorticoid Responsive Gene That Modulates Cytokine Function in Airway Smooth Muscle Cells
Total Page：16
File Type：pdf, Size：1020Kb
RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Himes, B. E., X. Jiang, P. Wagner, R. Hu, Q. Wang, B. Klanderman, R. M. Whitaker, et al. 2014. “RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells.” PLoS ONE 9 (6): e99625. doi:10.1371/journal.pone.0099625. http:// dx.doi.org/10.1371/journal.pone.0099625. Published Version doi:10.1371/journal.pone.0099625 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:12406565 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells Blanca E. Himes1,2,3*., Xiaofeng Jiang4., Peter Wagner4, Ruoxi Hu4, Qiyu Wang4, Barbara Klanderman2, Reid M. Whitaker1, Qingling Duan1, Jessica Lasky-Su1, Christina Nikolos5, William Jester5, Martin Johnson5, Reynold A. Panettieri Jr.5, Kelan G. Tantisira1, Scott T. Weiss1,2, Quan Lu4* 1 Channing Division of Network Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, United States of America, 2 Partners HealthCare Personalized Medicine, Boston, Massachusetts, United States of America, 3 Children’s Hospital Informatics Program, Boston, Massachusetts, United States of America, 4 Program in Molecular and Integrative Physiological Sciences, Departments of Environmental Health, and Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America, 5 Pulmonary, Allergy and Critical Care Division, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America Abstract Asthma is a chronic inflammatory respiratory disease that affects over 300 million people worldwide. Glucocorticoids are a mainstay therapy for asthma because they exert anti-inflammatory effects in multiple lung tissues, including the airway smooth muscle (ASM). However, the mechanism by which glucocorticoids suppress inflammation in ASM remains poorly understood. Using RNA-Seq, a high-throughput sequencing method, we characterized transcriptomic changes in four primary human ASM cell lines that were treated with dexamethasone—a potent synthetic glucocorticoid (1 mM for 18 hours). Based on a Benjamini-Hochberg corrected p-value ,0.05, we identified 316 differentially expressed genes, including both well known (DUSP1, KLF15, PER1, TSC22D3) and less investigated (C7, CCDC69, CRISPLD2) glucocorticoid- responsive genes. CRISPLD2, which encodes a secreted protein previously implicated in lung development and endotoxin regulation, was found to have SNPs that were moderately associated with inhaled corticosteroid resistance and bronchodilator response among asthma patients in two previously conducted genome-wide association studies. Quantitative RT-PCR and Western blotting showed that dexamethasone treatment significantly increased CRISPLD2 mRNA and protein expression in ASM cells. CRISPLD2 expression was also induced by the inflammatory cytokine IL1b, and small interfering RNA-mediated knockdown of CRISPLD2 further increased IL1b-induced expression of IL6 and IL8. Our findings offer a comprehensive view of the effect of a glucocorticoid on the ASM transcriptome and identify CRISPLD2 as an asthma pharmacogenetics candidate gene that regulates anti-inflammatory effects of glucocorticoids in the ASM. Citation: Himes BE, Jiang X, Wagner P, Hu R, Wang Q, et al. (2014) RNA-Seq Transcriptome Profiling Identifies CRISPLD2 as a Glucocorticoid Responsive Gene that Modulates Cytokine Function in Airway Smooth Muscle Cells. PLoS ONE 9(6): e99625. doi:10.1371/journal.pone.0099625 Editor: Jan Peter Tuckermann, University of Ulm, Germany Received December 6, 2013; Accepted May 17, 2014; Published June 13, 2014 Copyright: ß 2014 Himes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by National Institutes of Health (NIH) U01 HL65899, an NIH Pharmacogenomics Research Network (PGRN) – RIKEN Center for Genomic Medicine (CGM) Global Alliance, R01 HL097796, R01 HL114769, and P30 ES013508. BEH was funded by NIH K99 HL105663. XJ was supported by NIH training grant T32 HL007118. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors declare that no competing interests exist. * E-mail: [email protected] (BH); [email protected] (QL) . These authors contributed equally to this work. Introduction partly a result of 1) GC-GR complexes stimulating anti- inflammatory genes by directly binding to DNA at glucocorticoid Asthma, a chronic inflammatory respiratory disease that affects receptor enhancer elements, and of 2) GC-GR complexes over 25 million Americans and 300 million people world-wide, is inhibiting proinflammatory transcription factors such as nuclear characterized by variable airflow limitation and airway hyperre- factor kappa-light-chain-enhancer of activated B cells (NFkB) [6]. sponsiveness [1,2]. Glucocorticoids (GCs) are common medica- In addition to directly reducing inflammation, GCs have been tions used to treat various inflammatory diseases, including asthma shown to affect other asthma-related phenotypes, including [3]. Inhaled corticosteroids, GC medications that act directly in bronchodilation [7], airway hyperresponsiveness [8], and airway the lung, are among the most common asthma controller smooth muscle (ASM) contractility [9]. medications and treatment of asthma patients with them leads to Many cells and tissues are involved in asthma and are targeted improved clinical outcomes, including decreased asthma symp- by GCs, including inflammatory [10,11], airway epithelium [12], toms and exacerbations [4]. At a cellular level, GCs act by binding and ASM [13]. Of these, the ASM is involved in altered airway to GC receptors (GRs), causing them to translocate to cell nuclei contractility [14], a major asthma-specific trait that is assessed where they modulate transcription of various genes in a tissue- clinically and for research studies by measures such as broncho- dependent fashion [5]. The anti-inflammatory action of GCs is dilator response [15] and airway hyperresponsiveness [16]. PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e99625 CRISPLD2 Is a Glucocorticoid Responsive Gene in ASM However, compared to the other airway cells, much less is known Overall, 316 genes were significantly differentially expressed about how GCs work specifically in the ASM to alleviate asthma. after correcting for false discovery rate by the Benjamini- Because GCs function by activating GR to directly modulate Hochberg [23] approach [Figure 1A, Table S3]. Table 1 contains transcriptional gene expression, a better understanding of how the the genes with Q-value ,1E-10 that were considered for further ASM transcriptome responds to GCs is needed to provide study. Some of these top genes have been previously related to mechanistic insights for improving asthma therapy. Several studies steroid responsiveness and inflammation (i.e., DUSP1 [24], FKBP5 have been conducted to identify GCs-induced transcript changes [25], KLF15 [17], PER1 [12,26], and TSC22D3 [25,27]), and their in the ASM. For example, two microarray-based gene expression upregulation by 1 mM for 18 DEX was confirmed by quantitative studies have measured the effect of GCs on ASM cells using in vitro real time PCR (qRT-PCR) in ASM cells from three donors models where human ASM cells were stimulated with dexameth- [Figure 1B]. qRT-PCR results for the fourth donor used in the asone or fluticasone [17,18]. Although both were limited by the RNA-Seq experiment were also consistent [Figure S2]. Other inherent biases of microarrays, these studies identified some genes genes identified via the RNA-Seq experiment were considered involved in the ASM GC response, with one focusing on validating potentially novel GC-responsive genes as they have little published the function of the KLF15 gene in airway hyperresponsiveness [17] evidence regarding a relationship with steroid responsiveness and/ and the other on the overlap between GC and beta-agonist or inflammation. Gene set enrichment analysis using the NIH response of the ASM [18]. DAVID tool [28] identified various Gene Ontology and other Recent advances in sequencing technologies have made possible annotation categories that were overrepresented by the 316 genes. the comprehensive and in-depth characterization of transcrip- The top six gene functional annotation clusters (enrichment scores tomes via a technique known as RNA-Seq [19–21]. Compared to .3) had terms related to: glycoprotein/extracellular matrix, the use of microarrays, RNA-Seq is able to (1) quantify more RNA vasculature development, circulatory system process, response to species, including non-coding and novel splice variants, (2)